بومشناسی جنگلهای ایران (May 2024)
Optimization of in Vitro Cultivation Conditions of Blood Hawthorn (Crataegus atrosanguinea)
Abstract
Extended Abstract Background: It is important to research in the field of germplasm protection of native and medicinal tree species in northern Iran forests, especially blood hawthorn with many medicinal properties. Sexual reproduction of hawthorn is possible with apomixis due to the thick shell and two types of physical and physiological dormancy, and its asexual reproduction by rooting cuttings faces many problems. Bloody hawthorn (Crataegus atrosanguinea from the Rosaceae family) is considered one of the valuable species of the forests of the north and west of the country, which is very important due to its fruitfulness, very strong wood, and numerous medicinal properties. The medicinal properties of hawthorn have been proven in the treatment of blood pressure, arteriosclerosis, heart congestion, heart muscle weakness, and coronary insufficiency. Hawthorn contains many secondary metabolites such as flavonoids proanthocyanidin and glycosides. Optimizing the in vitro cultivation conditions of blood hawthorn as one of the natural species of Iran can be a suitable solution for its propagation. The present research was carried out to control pollution, control browning, and induce the callus of blood hawthorn (C. atrosanguinea) in vitro. Controlling the contamination of explants, seeds, and plant tissues is of great importance for the success of plant tissue culture. Since the enrichment of natural plant explants is a priority for tissue culture, this is usually associated with many problems with browning, especially for woody plants that have a thick and woody stem. Determining the type and concentration of the growth regulator of a variable factor is necessary for the growth of plants under in vitro conditions. After applying the growth regulator to the culture medium, the plant will show different reactions, but one of the appropriate ways to choose the correct path of plant regeneration is to determine the wet and dry weights of the callus, which is widely measured as a standard of callus growth. The callus growth curve can be obtained using the wet and dry weights of the callus. The results of past research indicate a wide range of changes in the type and concentration of growth regulators on callus formation and regeneration of different species of the hawthorn genus. A specific type and concentration of growth regulators should be applied according to the type of plant species. Methods: Young and healthy stems of blood hawthorn were collected from Larijan in Amol, Mazandaran province, Iran, with geographical coordinates of 38' 58° N and 10' 52° E, at the beginning of the growing season. The collected stems were immediately transferred to the plant tissue culture laboratory of the Faculty of Natural Resources at Sari University of Agriculture and Natural Resources, Iran. Disinfection of natural hawthorn stems is very necessary due to surface and tissue contamination. For this purpose, explants of 0.5-1 cm size were cut from the stems. The infection control treatments included HgCl2, H2O2, NaClO, and AgNO3, and five treatments were designed to control the browning of explants. Browning control treatments included control, running water, ascorbic acid, polyvinyl chloride, and activated charcoal. In general, the culture medium consisted of substances including mineral salts (micro- and macroelements) as providers of maximum growth, amino acids and vitamins as nitrogen sources, sugar as a source of carbon and energy, growth regulators as growth and morphogenesis stimulants, agar as the solidifier of the cultivation medium, and water, which includes 90% of the cultivation medium. In this research, the culture medium was MS, which was first created by Driver and Koniaki (1984) and prepared according to Dufussard (1976). In this study, the pH of the culture medium was set to 5.8. For callus induction, IBA, NAA, 2,4-D, BAP, TDZ, and Kin treatments at three concentration levels of 0.1, 1, and 6 mg/L and a combination of NAA (6 mg-1) + BAP (1 mg-1), and IBA (6 mg l-1) + BAP (1 mg-1) were used in the MS culture medium. Stem explants were placed horizontally on the culture medium. Among different callus treatments, callus percentage, degree of callus, callus fresh and dry weights, and cross-sectional area of callus were measured to investigate callus induction. The best treatment was obtained by calculating the mentioned characteristics and that treatment was used for planting. It was transplanted approximately every 20 days and a total of 5-6 times. After callus cultivation in the reproduction medium, the explants were placed in a culture chamber and adaptation racks under 16 hours of light and 8 hours of dark treatment at 25 ± 2 °C with a humidity of 70%. Results: HgCl2 (0.5%, 16 minutes) with an average of 100% health was the most appropriate pollution control treatment. The control treatment with 98% health was observed as the most appropriate browning control treatment. Moreover, the type, concentration, and interaction effect of the type and concentration of the growth regulator significantly affected the callus induction characteristics of blood hawthorn (P < 0.01). The highest fresh and dry weights of callus, percentage and degree of callus formation, and cross-sectional area of callus were observed among auxin and cytokinin treatments, respectively, for 6 mg/L of NAA and 1 mg/L of BAP treatments. The highest levels of the studied callus induction characteristics were observed in the culture medium containing the combination of auxin and cytokinin (6 mg-1 of IBA+1 mg-1 of BAP). Conclusion: In general, the results showed a 90% chance of controlling the browning of the explants of natural blood hawthorn branches in a normal state and without applying any treatment. It is recommended to use 0.5% mercuric chloride for 16 minutes to disinfect the explants. The combination of growth regulators in the culture medium was evaluated as very suitable for the callus induction of bloody hawthorn. The results of this research can provide laboratory instructions for the regeneration and mass reproduction of blood hawthorn.
